PROJECT SUMMARY Of the greater than 6 million fractures occurring yearly in the US, 5-20% will result in nonunion or delayed union. Cell based therapies represent an exciting alternative to traditional bone grafting or implants, but cell transplantation requires a tailorable substrate to provide necessary cues to implanted cells. Mesenchymal stem/stromal cells (MSCs) are an attractive cell source for use in tissue engineering because of their robust secretion of proangiogenic and anti-inflammatory trophic factors. Upon appropriate stimulation, MSCs can directly contribute to bone formation by differentiating to bone-forming osteoblasts, yet osteogenically induced MSCs suffer from reduced secretion of proangiogenic factors. We demonstrated that the presentation of a proangiogenic peptide, Gly-His-Lys (GHK), to MSCs entrapped in alginate hydrogels resulted in up to a 4-fold increase in their proangiogenic potential. We previously incorporated peptide ligands such as Arg-Gly-Asp (RGD) to facilitate cell adhesion to ionically-crosslinked alginate and photocrosslinkable alginate gels (PAHs) with more controlled degradation profiles. RGD stimulates osteogenic differentiation of MSCs but may impair secretion of endogenous proangiogenic cues. Thus, there is a pressing need for biomaterials that can simultaneously enhance the proangiogenic and osteogenic potential of transplanted MSCs to maximize their efficacy in cell based therapies. Our central hypothesis is MSCs can be simultaneously stimulated to undergo osteogenic differentiation while secreting potent proangiogenic cues, translating to enhanced therapeutic potential by increasing local vascularization and bone formation. Aim 1. Determine the role of dual peptide signaling on MSC osteogenic differentiation and proangiogenic potential when entrapped in PAHs. We will synthesize PAHs with varying densities of RGD and GHK. Changes in the biophysical properties of the gel, as well as the osteogenic and proangiogenic response of entrapped human MSCs will be determined. Aim 2. Define the necessary biophysical properties of peptide-presenting PAHs to instruct MSC osteogenic and proangiogenic potential. We will examine the role of each peptide on osteogenic differentiation and proangiogenic potential, while measuring the contributions of cell adhesion and substrate bulk stiffness to MSC response. Aim 3. Demonstrate the therapeutic potential of MSCs deployed in dual peptide-modified alginate to promote vascularization and bone repair in rodent critical-sized calvarial bone defects. We will characterize the capacity of MSCs implanted in peptide-presenting PAHs to promote bone repair in an orthotopic defect. The role of implanted cells, quantity, and quality of bone formation will be assessed using noninvasive imaging modalities and histological analysis. The proposed research is innovative because it exploits the activity of two distinct peptides with a biodegradable hydrogel to potentiate the reparative potential of MSCs. This research will provide a novel approach for regulating bone formation, and the strategies have potential in enhancing the efficacy of materials-based therapies for tissue repair.